Apparatus for multi-zone polymerase chain reaction

ABSTRACT

A process of amplifying a nucleic acid sequence by a procedure involving a polymerase chain reaction or a ligase chain reaction. The process involves repeated cycles of steps including a nucleic acid denaturing step and a nucleic acid synthesis step, the synthesis step being carried out under the action of an enzyme (a nucleic acid polymerase or ligase). The denaturing step and the synthesis step are carried out in different denaturing and synthesis reaction zones, respectively, and, during the repeated cycles, the enzyme is maintained in isolation from the denaturing reaction zone, and conditions or reagents required for the denaturing step are maintained in isolation from the synthesis reaction zone to the extent that the reagents and conditions required for denaturing do not impede the synthesis reaction to a substantial extent. The use of separate zones for the steps of the reactions means that an enzyme that is destroyed or degraded by the reagents and conditions required for denaturing (e.g. a thermolabile or alkalolabile polymerase or ligase) may be used in the reaction. Moreover, the use of multiple zones means that inexpensive equipment may be used for the process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of our prior application Ser. No.09/035,091 filed Mar. 5, 1998, now U.S. Pat. No. 5,912,129.

BACKGROUND OF THE INVENTION

I. Field of the Invention

This invention relates to techniques used for the polymerization andamplification of sequences of nucleic acids of prokaryotic andeukaryotic origin involving the polymerase chain reaction or the ligasechain reaction. More particularly, the invention relates to a processand apparatus for carrying out such techniques.

II. Background Art

The amplification of nucleic acid sequences by means of a chain reactiontechnique using a nucleic acid polymerase enzyme has been developed overthe last several years to enable minute amounts of nucleic acids, e.g.DNA, to be copied in quantity to an extent suitable for detection andanalysis. The procedure is described, for example, in U.S. Pat. Nos.4,683,195, 4,683,202, 5,436,149, 5,405,774, 5,340,728, 5,338,671 and4,965,188, the disclosures of which are incorporated herein byreference. Recently, a related chain reaction technique involving anucleic acid ligase enzyme has also been developed.

The polymerase chain reaction (PCR) procedure is briefly explained asfollows. In nature, the replication of duplex DNA is carried out by thekey enzyme DNA polymerase that has two activities, namely:

a) 5'-3' polymerase activity, i.e. the addition of new nucleotides tothe growing strand at the 3-prime end of a primer, probe (labeledprimer) or synthesizing strand; and

b) 3'-5' exonuclease activity, i.e. the removal of nucleotides from the3-prime end which may be misincorporated into the synthesizing strand.

The enzyme can be used for artificial replication of DNA by employingfour basic elements:

a) A single stranded DNA or RNA template.

b) An oligonucleotide (primer or probe) having a nucleotide at the3'-prime end carrying a hydroxy group at the 3rd position of the sugarmolecule and carrying a base molecule which is complementary to thecorresponding base molecule on the single stranded template strand.

c) A set of synthetic nucleotides (dTTP, dCTP, dGTP, dATP) activated bymagnesium ions at pH 8.3-pH 8.4.

d) A DNA polymerase enzyme which has the ability to add new nucleotidesat the 3-prime end of the primer (or to the growing chain) viaphosphodiester bonds.

Given the fact that DNA occurs naturally as two complementary strandsjoined by hydrogen bonds, and that both strands of DNA may function astemplates for the synthesis of new strands by polymerization, it ispossible to duplicate a specific segment of DNA by using a pair ofcomplementary oligonucleotides as primers which can bind across (i.e. onopposite sides of) the segment of interest (the so-called targetsequence). It is known that, to facilitate the binding of primers to thetemplate, the hydrogen bonds across the double stranded DNA first haveto be broken and the single strands have to be separated.Conventionally, this is carried out by heating the template to atemperature greater than 94° C. and, when the sample cools, allowing theoligonucleotide primers to bind to (anneal with) complementary regionson the template. This is followed by the polymerase reaction whichcauses new double stranded DNA to be formed by nucleotide polymerizationusing the target sequence as a template for the selection of nucleotidesfor the new complementary sequence. The polymerase chain reactionextends this concept by making more copies of the target sequence byrepeating the sequence of steps by cycling the temperature around thedenaturing point while maintaining the reactants in a single chamber orreaction zone. Since normal DNA polymerase is heat sensitive (i.e.thermolabile), and is deactivated if heated to the temperature requiredfor the separation of strands of DNA, it was initially necessary to addan influx of fresh polymerase prior to the synthesis step for eachcycle. To overcome this limitation, a heat stable polymerase wasisolated from Thermophilus acquaricus. Recently, the gene forthermostable polymerase has been cloned in expression vectors, and arecombinant heat stable enzyme produced and made commercially available.

The most common heat stable enzyme of this type is referred to as TaqDNA polymerase from Thermophilus acquaticus, but others are also known,e.g. Tth DNA polymerase from Thermophilus thermophiles and Tth DNApolymerase from Thermophilus flavus. The existence of such enzymes makesit possible to combine the starting materials and reactants in a singlereaction zone or chamber and to cycle the temperature above and below94° C. to produce the steps required for PCR without further additionsof polymerase.

Thus, at present, the polymerase chain reaction in its most common formhas three stages. They are:

a) Denaturing--Denaturing is a process whereby the individual strands ofthe DNA are separated by breaking the hydrogen bonds across the bases ofthe complementary nucleotides. At present, this is normally achieved byheating the DNA to near the boiling point of water (more specifically,to a "melting" temperature greater than 94° C.).

b) Annealing--This is a process whereby synthetic oligonucleotideprimers or probes (each normally containing about 20 nucleotides) bindto complementary sequences of any single DNA strand present by theformation of hydrogen bonds across the bases of the complementarynucleotides. Pairs of primers are generally used, one for each strand ofthe DNA, flanking a sequence of interest, normally about 100 to 5,000base pairs (bp) in length. The temperature at which annealing takesplace is normally 37° to 70° C.

c) Polymerization--This involves the addition of new nucleotides at the3'-prime end of the primer by the formation of phosphodiester bonds inthe presence of Taq DNA polymerase. The polymerization reaction normallytakes place at a temperature of about 72° C.

When this cycle is repeated many times (normally at least 30 times witheach cycle typically lasting from 3 to 5 minutes), a detectable amountof the target DNA is produced.

The ligase chain reaction (LCR) is similar to PCR, except that shortstretches of nucleic acid (probes), bound to a target sequence template,are joined together by a nucleic acid ligase enzyme. LCR is often usedto distinguish between normal DNA of known sequence and mutant DNA. Apair of DNA probes having a sequence which, taken together, arecomplementary to the expected target nucleic acid are produced andbrought into contact with the target sequence in the presence of anucleic acid ligase enzyme. If double stranded target DNA is denaturedand allowed to anneal with the probes, the probes will bind to thetarget DNA with ends adjacent to each other. The ligase enzyme thenbinds the probe DNA to form a single strand comprising both probes. Ifthe target DNA differs from the expected sequence, the probes will notbind properly and the ligase enzyme will not be able to form thecombined single strand. Repeated cycles amplify the combined singlestrand (if formed) which can be distinguished from the probes themselvesby nucleotide length and by the presence of markers from both probes. Ifone of the probes is bound to a solid support and the other is not, thecombined single strand will also be bound to the solid support and,after washing to remove the unbound probe, the presence of the combinedstrand can be detected by the presence of the marker used for theunbound probe. As in the case of PCR, LCR is carried out by repeatedthermal cycles to cause denaturing and annealing of the DNA, so athermostable ligase enzyme is required.

In spite of the feasibility of PCR and LCR, the widespread use of thesetechniques has been limited somewhat by the high capital cost of therequired thermocycler apparatus, which tends to be complex in design andconstruction, and the high cost of the available thermostablepolymerases or ligases. Therefore, current PCR technology oftennecessitates centralized testing at sites which are often distant fromthe point of sampling. This arrangement not only increases the cost butalso delays the reporting of results. It would therefore be desirable toprovide a PCR method and apparatus that could be used less expensivelyand made more widely available.

SUMMARY OF THE INVENTION

An object of the invention is to make PCR and/or LCR testing andsynthesis less expensive and more readily available to researchers,medical personnel, manufacturers, and other potential users of thetechnology.

Another object of the invention is to enable PCR and LCR techniques tobe carried out in a practical manner using relatively inexpensivepolymerase and ligase enzymes.

Another object of the invention is to provide a PCR or LCR method thatis not reliant on thermostable enzymes and that can be carried out infairly simple apparatus that can be provided at or close to the sourceof sampling or use of the DNA.

Yet another object of the invention is to simplify apparatus used forcarrying out nucleic acid amplification by the polymerase chain reactionor the ligase chain reaction.

According to a first aspect of the present invention, there is provideda process of amplifying a nucleic acid sequence by a procedure selectedfrom the group consisting of a polymerase chain reaction and a ligasechain reaction, involving repeated cycles of steps including a nucleicacid denaturing step and a nucleic acid synthesis step, said synthesisstep being carried out under the action of an enzyme, wherein thedenaturing step and the synthesis step are carried out in differentdenaturing and synthesis reaction zones, respectively, and wherein,during said repeated cycles, the enzyme is maintained in isolation fromthe denaturing reaction zone, and conditions or reagents required forthe denaturing step are maintained in isolation from the synthesisreaction zone to the extent that said reactions and conditions do notimpede said synthesis reaction substantially. According to a secondaspect of the invention, there is provided an apparatus for carrying outthe above process, the apparatus comprising a container for reactantsrequired during said denaturing step, a container for reactants requiredduring said synthesis step, including said enzyme, a solid phase supportfor binding nucleic acid formed during said synthesis step, and meansfor separately and successively contacting said solid support with saidreactants required during said denaturing step and said reactantsrequired during said synthesis step.

According to a third aspect of the invention, there is provided acassette unit for carrying out a process involving amplifying a nucleicacid sequence by a procedure selected from the group consisting of apolymerase chain reaction and a ligase chain reaction, involvingrepeated cycles of steps including a nucleic acid denaturing step and anucleic acid synthesis step, said synthesis step being carried out underaction of an enzyme, wherein the denaturing step and the synthesis stepare carried out in different denaturing and synthesis reaction zones,respectively, and wherein, during said repeated cycles, the enzyme ismaintained in isolation from the denaturing reaction zone, andconditions or reagents required for the denaturing step are maintainedin isolation from the synthesis reaction zone to the extent that saidreactions and conditions do not impede said synthesis reactionsubstantially; said cassette unit comprising an enclosed housing havingat least two chambers for holding liquid reactants and maintaining saidliquid reactants separate from each other and a solid support fornucleic acid provided in one said container, said housing including atleast one passage allowing said solid support to be brought successivelyand temporarily into contact with said liquid reactants from said atleast two containers.

By the term "reaction zone" as used herein we mean a region of reactantsand conditions that result in a desired reaction (denaturing, synthesis,etc.). The regions forming the denaturing zone and the synthesis zonemay be separated by distance (e.g. they may be located in differentcontainers) or simply by time (e.g. the zones may be physically locatedin the same container but the reactants and conditions may vary overtime to bring about different reactions).

By isolating the polymerase or ligase enzyme (hereinafter simply the"polymerase" or the "ligase") from the conditions or reactants requiredin the denaturing zone, an enzyme that is destroyed or deactivated bythose reactants or conditions may be employed in the invention.Furthermore, by substantially isolating the conditions or reagentsrequired for the denaturing zone from the synthesis zone, synthesis mayproceed without any tendency of the newly-forming nucleotide polymer toseparate from the template sequence. The nucleotide sequences aretherefore cycled between the denaturing zone and the synthesis zonewhile other reagents and conditions required in those zones are kept inisolation from each other.

The conditions or reagents required for the denaturing step may beelevated temperature (above 94° C.) or aqueous alkali, although anyother method of denaturing double stranded DNA may be used.

The polymerase or ligase used in the present invention is preferably athermolabile (heat sensitive) enzyme because such enzymes are usuallyfar less expensive than thermostable (heat tolerant) enzymes. Thepresent invention makes it possible to use a thermolabile polymerase orligase even if elevated temperature is used to bring about thedenaturing step, since the enzyme is not exposed to the elevatedtemperature. However, a thermostable enzyme may alternatively be used inthe present application if an inexpensive enzyme of this type can befound, or if the expense of the enzyme is acceptable to the user. Evenwhen an expensive thermostable polymerase or ligase is used, the presentinvention has the advantage that the reaction can be carried out insimple and inexpensive apparatus.

Suitable thermolabile polymerases for use in the present inventioninclude the following:

DNA polymerase 1 E. coli lambda lysogen NM 864;

DNA polymerase 1 Large Klenow fragment which lacks 5'-3' exonucleaseactivity;

AMV reverse transcriptase;

Murine reverse transcriptase;

M-MLV (Moloney murine Leukemia virus) reverse transcriptase expressed inE. coli as pol gene;

SP6 RNA polymerase;

T3 RNA polymerase;

T7 RNA polymerase;

T4 DNA polymerase; and

T7 DNA polymerase.

The above enzymes are all commercially available (e.g. from LifeTechnologies, Gaithersburg, Md., USA; and Amersham Life Science Inc.,Canada). Of course, other polymerase enzymes may be employed, includingchimeric recombinant enzymes carrying a polymerase moiety.

Examples of thermostable polymerases have been mentioned above.

Suitable thermolabile ligases include the following:

E. coli DNA ligase,

T4 DNA ligase, and

T4 RNA ligase.

The above ligases are commecially available, for example, fromBoehringer Mannheim, Canada.

Examples of thermostable ligases include the following:

recombinant pfu DNA ligase from Pyrococcus furiosus.

The above ligase is commercially available from Stratagene, USA.

When the denaturing step is brought about by subjecting the doublestranded DNA to aqueous alkaline conditions, i.e. by elevating the pH ofthe DNA-containing solution in the denaturing zone, elevatedtemperatures can be avoided. Most conventional polymerases and ligasesare destroyed or deactivated by exposure to alkali, so again the presentinvention makes it possible to use a conventional inexpensive polymeraseor ligase in the process since the enzyme is isolated from thedenaturing conditions. In practice, however, it is preferable to use analkali-resistant (alkalophilic) polymerase or ligase because some smallamounts of the alkali may be carried over from the denaturing step tothe synthesis (polymerization/ligation) step together with the denaturedDNA. However, the amount of alkali carried over in this way must not beenough to interfere to any significant extent with thepolymerization/ligation reaction, so there is still substantialisolation of the alkali from the polymerization/ligation zone despitesome minor carry over.

Alkali tolerant polymerases and ligases may be obtained fromalkali-tolerant microorganisms as disclosed, for example, inAlkalophilic Microorganisms, by Koki Horikoshi and Teruhiko Akiba, JapanScientific Press, Tokyo, 1982, the disclosure of which is incorporatedherein by reference. Examples of such alkali tolerant microorganismsinclude the following:

Bacteria: Bacillus subtilis

Blue green algae: Plectonema nostocorum Arthrospira plantenesis

Fungi: Penicillium variables Fusarium bullatum Fusarium oxysporium.

A known alkali-tolerant RNA polymerase suitable for use in the inventionis obtained from Bacillus subtilis, and has an operable pH range of7.0-9.3 with a maximum activity at pH 8.0.

The polymerase/ligase may be isolated from the denaturing step in anysuitable way and the conditions or reagents required for the denaturingstep may be isolated from the polymerization/ligation zone in anysuitable way. This requires separating the polymerase/ligase from thenewly formed double stranded DNA so that the DNA may proceed to thedenaturing zone, and then removing the newly formed single stranded DNAfrom the denaturing zone so that it may proceed to the synthesis(polymerization/ligation) zone.

Most conveniently, the nucleic acid and/or the primers and/or the probesare immobilized on a solid support and the solid support is thensuccessively and temporarily contacted with reagents and conditions thatare appropriate for each step of the process, thus establishingdifferent reaction zones. The immobilized nucleic acid can be easily andquickly contacted with and then removed from liquid reactants or regionsof elevated temperature, i.e. from one reaction zone to another, so thatthe PCR or LCR cycles may be carried out rapidly and conveniently. Thus,the solid support may be movable and may be immersed successively intodifferent reaction containers holding the required reagents andproviding the required conditions of temperature or alkalinity, oralternatively the solid may be fixed and held in a single container andthe required reagents for each step successively and temporarilyintroduced into the container and the appropriate conditions (e.g.temperature) applied. Thus, the nucleic acid can be separated from thepolymerase/ligase after the polymerization/ligation reaction, prior tothe denaturing reaction of the next cycle, and then the single strandednucleic acid produced by the denaturing reaction can be contacted withthe reactants and polymerase/ligase prior to the nextpolymerization/ligation reaction.

In a preferred form, the invention provides a process of amplifying anucleic acid sequence, including repeated cycles of the steps ofdenaturing of complementary strands of nucleic acid, annealing of saidnucleic acid with complementary primers flanking a target sequence, andDNA polymerase catalyzed polymerization of a sequence of nucleic acidcomplementary to said target sequence extending from said primers usingthe target sequence as a template, which process comprises: providingseparate reaction zones having conditions appropriate for each of saiddenaturing, annealing and polymerization steps, and exposing saidnucleic acid and synthesized nucleic acid sequences repeatedly to saidreaction zones in an order and with residence times in each zoneappropriate for effecting a polymerase chain reaction, wherein saidreaction zone for said synthesis step contains a thermolabile DNApolymerase and has a temperature suitable for polymerization of nucleicacids that remains below a temperature at which activity of saidpolymerase is adversely affected.

Thus, the original and synthesized nucleic acid may be passed repeatedlythrough the reaction zones by immobilizing the nucleic acid on a solid,but easily-transportable support acting as a transport medium and movingthe solid support from one reaction zone to another. The solid supportmay be, for example, a magnetic substrate (preferably in the form ofbeads) coated with anti-DNA antibody, or a type of paper that may bindDNA directly. When the solid transport medium is magnetic, the solidsupport medium may be moved through the zones by causing the transportmedium to follow movements of a magnetic element (introduced directlyinto the zones or positioned immediately outside a container enclosingthe reaction zones).

According to another preferred form of the invention, there is providedan apparatus for amplifying a nucleic acid sequence by a processinvolving repeated cycles of denaturing double stranded nucleic acid toform separated single strands, annealing the single strands of nucleicacid with at least one complementary primer or probe flanking a targetsequence, and, using the target sequence as a template extending fromthe primers or probe, forming a complementary sequence of nucleic acidsby nucleic acid polymerization in the presence of a nucleic acidpolymerase enzyme to form double stranded nucleic acid, said apparatuscomprising a container for reactants required during said denaturingstep, a container for reactants required during said polymerizationstep, including said nucleic acid polymerase enzyme, a solid phasesupport for binding nucleic acid formed during said polymerization step,and means for separately and successively contacting said solid supportwith said reactants required for during said denaturing step and saidreactants required during said polymerization step.

According to a further preferred form of the invention, there isprovided apparatus for carrying out a process of amplifying a nucleicacid sequence, involving repeated cycles of the steps of denaturing ofcomplementary strands of nucleic acid, annealing of said nucleic acidwith complementary primers flanking a target sequence, and DNApolymerase catalyzed polymerization of a sequence of nucleic acidcomplementary to said target sequence extending from said primers usingthe target sequence as a template polymerization, said apparatuscomprising: a plurality of containers for containing liquids formingreaction zones for denaturing, annealing and polymerization of nucleicacids, a solid support for immobilizing said nucleic acid andsynthesized nucleic acid, and means for moving said solid support fromone container to another in a sequence appropriate for effecting apolymerase chain reaction, wherein at least a container provided forsaid synthesis step is provided with means to prevent a temperature insaid container from reaching a temperature at which activity ofthermolabile DNA polymerase held in said container is destroyed.

In the present invention, the original nucleotide sequence intended foramplification may be, for example, genomic DNA, DNA/RNA duplexes orcDNA. The method is also applicable to asymmetric amplification usingeither single stranded DNA or RNA as a template (a process carried outto obtain sufficient copies of a single stranded nucleic acid sample fornucleotide sequencing or appropriate testing). The invention is alsoapplicable to methods of detection or confirmation based on LCR.

This technology has application in the following areas:

a) Identification of microbial pathogens.

b) Identification of mutations and hereditary diseases.

c) Quantification of gene expression in cells.

d) DNA sequencing.

e) RNA sequencing.

The advantages over the conventional amplification using thermostablepolymerase/ligase are:

a) There is no need for an expensive thermocycler (the apparatusconventionally used for the PCR/LCR procedure) and therefore therequired capital cost is less. This may make the equipment sufficientlycost effective that the process may carried out in a decentralizedtesting facility (such as doctors' offices).

b) The apparatus may be designed to hold one or more removable housings(referred to herein as cassettes) each enclosing multiple containers forthe various steps of the PCR/LCR method. When multiple cassettes areused, it is possible to carry out two or more PCR/LCR reactions ondifferent substrates simultaneously, e.g. to analyze more than one typeof sample at any one time on the same equipment using differentcassettes, e.g. E. coli and Salmonella. Such cassettes may be producedas disposable units that may be kept sealed and provided with sealableinlets/outlets.

c) The number of samples analyzed at any one time can be varied. Theequipment may have capacity to deal with:

1-5 samples at a time

1-10 samples at a time

1-25 samples at a time

1-50 samples at a time

1-100 samples at a time

1-200 samples at a time, or continuous feeding of cassettes.

The invention is described in more detail below with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating the various alternative PCRprocedures of the present invention and, in one case, the prior art;

FIG. 2 is a flow chart similar to FIG. 1 illustrating the various LCRprocedures of the present invention and, in one case, the prior art;

FIG. 3 is a further flow chart illustrating a PCR procedure in moredetail;

FIG. 4 shows an example of a magnetic particle of the type that may beused as a transport medium in the process of the present invention;

FIG. 5 is a diagram showing the scheme of a single primer polymerasechain reaction according to a preferred embodiment of the invention;

FIGS. 6a, 6b, 6c and 6d are diagrammatic representations of a preferredform of the process and apparatus of the present invention duringvarious steps of the process;

FIG. 7 is a top plan view of a holder apparatus for a number ofcassettes of the type shown in FIGS. 6a-6d;

FIG. 8 is a diagrammatic representation similar to FIG. 6a of anapparatus which makes use of a paper-type transport medium rather thanmagnetic beads; and

FIG. 9 is a diagrammatic representation of a further apparatus in whichthe nucleic acid is bound to a stationary (anchoring) medium andtreatment solutions are successively brought into contact with thestationary medium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention operates in essentially the same way for LCR andfor PCR, i.e. by repeated denaturing, annealing and synthesis of nucleicacid. There are various alternative procedures for each type oftechnique. The alternatives available for PCR are shown in the flowchart of FIG. 1. The alternatives available for LCR are shown in theflow chart of FIG. 2.

Referring first to FIG. 1, following the illustrated flow chart from topto bottom, it will be apparent that the denaturing of the DNA may becarried out either by heat or by the use of alkali. In either case, theprocedure may be carried out either in a single container (by bindingthe DNA in place and successively forming different reaction zonesaround the DNA at different times) or in multiple containers (the DNA ismoved successively from one container to another, each container forminga different reaction zone). When heat is used to cause denaturing of theDNA in a multiple chamber technique according to the present invention,the polymerase enzyme may be either heat tolerant (thermostable) or heatsensitive (thermolabile). In both cases, the polymerase is isolated fromthe heat-denaturing step. In the case of a single chamberheat-denaturing technique, the conventional procedure (prior art)employs a heat tolerant polymerase and exposes the polymerase to theheat-denaturing step, whereas the present invention employs a heatsensitive polymerase and isolates the polymerase from the heatdenaturing step.

In the case of alkali denaturing, again there is a choice of a multiplechamber or single chamber technique. In the case of the single chambertechnique, an alkali tolerant polymerase may be employed, in which casethere is no need to isolate the polymerase from the denaturing step(although this may still be desirable). Alternatively, an alkalisensitive polymerase may be used by isolating the polymerase from thedenaturing step. In the case of a multiple chamber technique, again thepolymerase may be alkali-sensitive or alkali-tolerant. In both cases,steps are taken to isolate the polymerase from the denaturing step.

The alternatives shown for LCR in FIG. 2 are essentially the same, andthe figure is believed to be self-explanatory. The following discussionfocusses on PCR as an example, but may be applied in equivalent ways toLCR.

In preferred forms of the present invention, as it relates to PCR, thebasic alternative procedures involving denaturing by heat or by alkaliare outlined as follows:

a) The double stranded DNA is denatured by heat (at a temperaturegreater than 94° C.) in an aqueous medium in a first zone, then thedenatured DNA is preferably moved to a second zone of lower temperature(less than about 50° C.) for annealing in a separate container, and thenthe annealed DNA and primers are finally moved to a polymerization zonewhere the thermolabile polymerase enzyme is present. The newly formeddouble stranded DNA is then returned to the denaturing zone and theprocess repeated.

b) The DNA is denatured in an alkaline medium in a first zone, then theDNA is preferably moved to a second zone for annealing at neutral pH andspecific ionic concentrations, and then the annealed DNA and primers aremoved to the polymerization zone where the polymerase is present. Thenewly formed double stranded DNA is then returned to the denaturing zoneand the process repeated.

Of course, when it is stated that nucleic acid is moved from one zone toanother, it should be understood that this movement is "relative", i.e.it may be achieved by maintaining the nucleic acid stationary andforming different zones around the nucleic acid at different times, aswell as by moving the nucleic acid from one zone to another formed indifferent containers or chambers.

In either type of process, there must be at least two reaction zones orsolutions, one for the denaturing step and the other for thepolymerization (synthesis) step. The annealing step is generally carriedout in a separate reaction zone, but may alternatively be incorporatedinto the denaturing zone or the polymerization zone, according to theparticular type of reaction scheme involved. In some cases, particularlywhen alkaline denaturing is employed, it may be desirable to providefour zones or solutions to allow for a separate washing step between thedenaturing step and the annealing step. These various possibilities, forboth temperature-based denaturing and alkali-based denaturing, areoutlined in detail in the general flow chart of FIG. 3, which isbelieved to be self-explanatory.

Both these procedures (temperature denaturing and alkali denaturing) maybe carried out by conjugating (attaching) the original sample DNAtemplate (or the oligomer primers) to a suitable solid phase supportthat can act as a transport medium for moving the nucleic acid betweenreaction zones, or as an anchoring medium around which reaction zonescan be formed successively with time. The solid support may be, forexample, either an anti-DNA antibody-coated solid or a specialpaper-type matrix capable of directly binding the DNA, e.g. HY-BOND™nylon paper. Other materials that may be used as solid supports includenitrocellulose paper, glass milk, particles coated with DNA bindingproteins, DEAE-Sephadel™, DEAE-Sephadex™, DEAE-cellulose andpolystyrene. Use may also be made of the biotin/streptavidin bindingeffect.

When the solid support is to be used as a transport medium, i.e. it isto be used to move the nucleic acid from one container to another, it ispreferably produced in the form of magnetic particles having surfaceproperties suitable for binding the nucleic acid. By employing amagnetic particulate solid, the support and immobilized nucleic acid maybe transported by attracting the support to a magnet and moving themagnet to cause the solid to follow the magnet. Since magneticattraction is not prevented by interposing thin walls between the magnetand the attracted solid, the magnet may be positioned outside acontainer containing the solid. The solid can therefore be moved withinthe container without having to introduce any transport means into thecontainer itself.

Magnetic beads are presently the preferred transport medium. Such beadsmay be made of any magnetisable material, e.g. iron oxide (note:although the beads are described herein as "magnetic", the beads neednot themselves act as magnets--they merely need to be susceptible tomagnet attraction). The beads are preferably spherical and maypreferably vary in size from about 0.5 microns to 1.5 microns. However,the diameters of the beads may be increased to 1 mm or more so thatmovement of the beads from one zone to another may be made moreconvenient and reliable. An example of a magnetic bead suitable for usein the present invention is shown in FIG. 4. The bead 10 in thisembodiment is a siliconized iron oxide particle attached by a ligand 11to an oligomer (e.g. 40 mer) probe 12. The ligand 11 (e.g. an aminoconjugate or a carboxy conjugate) may be any organic compound that bindsto or may be conjugated to the bead. Alternatively, the siliconized ironoxide particle may be coated with streptavidin which will conjugate withbiotinylated DNA. As a further alternative, the siliconized iron oxideparticle may be coated with anti-DNA antibody (raised against purifiedDNA as well as native DNA) so that DNA binds to the antibody and henceto the particle. Similar binding methods may be used for other solidphase substrates.

FIG. 5 illustrates how the process of the invention, employing a bead ofthe type shown in FIG. 4, can be used for single primer polymerase chainreaction. In this case, an oligomer primer 15 is attached to a magneticbead 10. The primer is then annealed with a denatured sample 16 of a DNAtemplate and polymerization is carried out in the presence of athermolabile polymerase to form an extended DNA strand 17. In a furtherdenaturing step, the extended strand 17 is separated from the DNAtemplate strand 16. During further cycles, the template strand 16 goeson to create another amplified strand 17' in the same way during furthercycles and the extended strand 17 anneals to a labelled oligomer primer(probe) 18, which is extended into a labelled extended strand 19 uponpolymerization. This results in a considerable amplification of labelledtarget DNA strands suitable for sequencing or analysis.

FIGS. 6a-6d show an example of a preferred apparatus for carrying outthe process of the invention using alkaline denaturing, at variousstages in the process. As shown in FIG. 6a, the apparatus comprises ahousing 20 enclosing four chambers 21, 22, 23 and 24 forming reactioncontainers capable of enclosing reaction zones. The lower parts of thechambers are not in fluid communication with each other, although thecontainers share a common head space 25 above the normal upper level ofcontained liquids forming a passage that allows the access to eachchamber for reasons that will become apparent. Each chamber contains aliquid 21a, 22a, 23a and 24a suitable for carrying out a step in theprocess. Chamber 21 is for the step of denaturing double stranded DNA inalkaline conditions. Chamber 22 is a neutralising chamber containing anaqueous liquid for neutralising alkali carried over from chamber 21.This chamber is also preferably provided with a temperature controldevice (not shown, but see FIG. 7) to maintain the liquid in the chamberabove the annealing temperature of the DNA. Chamber 23 is an annealingchamber containing a solution of primers. This chamber is also providedwith a temperature control device (not shown) that causes the liquid inthe chamber to fall below the annealing temperature of the DNA, thusallowing annealing to take place. Chamber 24 is provided for thepolymerization reaction and holds a liquid containing a nucleic acidpolymerase that is preferably alkali-tolerant. Again, this chamber maybe provided with a heat control device that ensures that the temperatureof the liquid is suitable for polymerization but does not rise above amaximum temperature at which the polymerase remains active.

FIG. 6a shows a number of magnetic beads 10, preferably of the typeshown in FIG. 4, held within chamber 21. These beads are capable ofbinding and immobilizing DNA held within the chamber 21. The singlestranded DNA formed during the denaturing step (caused by the alkalineconditions) is thus immobilized on the surface of the beads 10.

As shown in FIG. 6b, after the annealing step in chamber 21 is complete(after a few minutes), the beads 10 are transferred from chamber 21 tothe neutralizing solution of chamber 22. This is achieved by moving amagnet 27 along an outside wall of the housing 20 from a positionadjacent chamber 21, over a division 26 between chambers 21 and 22 anddown to a position adjacent to chamber 22. The magnetic beads 10 insidethe housing 20 follow the movement of the magnetic element 27 outsidethe housing and are thus transferred to chamber 22. The requiredmovement of the magnet 27 is made possible by attaching the magnet to alower end of a holder 28 that slides at its upper end along a wire frame29 that is shaped to cause the magnet to follow the required movement.

After a period of time suitable for neutralization in chamber 22, thebeads may then be transferred by operation of the same magnetic into theannealing chamber 23 (see FIG. 6c), and then (after a suitable period oftime) to the polymerization chamber 24 (FIG. 6d). After polymerizationis complete, the beads may then be transferred by the magnet 27 directlyto the denaturing chamber 21 where the cycle may be commenced again.This latter step is possible because the magnet is made to movehorizontally only along the upper part of the frame 29, except whenmoving the beads vertically out of the polymerization chamber and intothe denaturing chamber, i.e. in such a way that the beads along theupper sidewall of the container without falling into any of theintervening chambers. In this way, any number of cycles may be repeateduntil suitable quantities of the synthesized DNA have been amplified, atwhich time the beads may be removed from the housing and the attachedDNA extracted.

Openings 30, 31, 32, 33 are provided in the upper wall 34 of the housing20 and each is closed by a self-sealing pierceable diaphragm 35.Solutions may be introduced into and aspired from the chambers of thehousing through such openings by piercing the diaphragm with a needle ofa hypodermic syringe or other insertion device (not shown) andintroducing or removing the solution. In this way, the interior of thehousing 40 may be kept sterile (i.e. uncontaminated with foreign DNA orRNA). The housing 20 may be supplied to the user with the necessaryreactants already present in the appropriate chambers. For example,these reactants may be provided as freeze dried powders adhering to thewalls of each container. Before the start of the process, measuredamounts (e.g. 25 μl portions) of sterile distilled water are added toeach chamber through the sterile openings. Once this has been done,undue tilting of the holder has to be prevented in order to avoid mixingthe solutions from the adjacent containers.

The housing 20 of FIGS. 6a-6d may take the form of a disposable sealedcontainer (referred to herein as a "cassette") provided with theillustrated four chambers, i.e. it may be a self-contained unit that maybe inserted into and removed from stationary base unit that has thenecessary drive and heating mechanisms, etc. Each cassette 20, which maybe completely removable from the stationary base unit, may be madeentirely of an inexpensive thin-walled material, e.g. thin plastics,that does not shield the interior of the cassette from the magneticfield produced by the magnet 27 located outside the cassette. An idealsize for such a cassette is 3 inches by 3 inches by 5 inches.

FIG. 7 is a top plan view of an embodiment of a stationary base unit 40(acting as a holder apparatus) for the cassettes 20. The apparatusconsists of a body 41 having an upper surface 42 provided with eightslots 43, each slot being dimensioned to receive a separate removablecassette 20 (one of which is shown in place in FIG. 7) of the type shownin FIGS. 6a-6d. Each end 44, 45 of the body is provided with a furtherslot 46 for receiving a magnet 27. A drive mechanism for the magnet(shown in FIGS. 6a-6d, but not shown in FIG. 7) moves the magnets backand forth in the slots 46 sliding on wire frames 29 (not shown in thisfigure, but see FIG. 6a) following the vertical and horizontal pathpreviously illustrated. The operation of the drive mechanism may beeffected manually by an operator, but is more preferably carried outautomatically by means of a motor (not shown) controlled by a timer (notshown) programmed to move the magnets in such a way as to move the beads10 from one chamber of each cassette to another after a suitable periodof residence in each chamber (usually 30 seconds to 2 minutes).

If necessary for more reliable operation, each of the cassette slots 43may be provided with a pair of adjacent magnet slots 46 to ensure thatthe magnetized beads in each cassette will be exposed to a strongmagnetic field.

Each slot 43 may be provided with a heating element 47, positioned tolie adjacent to an appropriate chamber of a cassette 20, and atemperature sensing device 48 (e.g. a thermocouple) to measure thetemperature of the an adjacent chamber of the cassette. The temperaturesensing device and heater may be electronically linked to a power source(not shown) for the heater to turn the heater on or off according topredetermined temperatures sensed by the temperature sensor. For theembodiment employing denaturing by alkali, all of the chambers of all ofthe cassettes may be kept at the same specific temperature, although thedenaturing chamber 21 is preferably heated to an elevated temperature(but below the heat-denaturing temperature) to assist denaturing of theDNA (denaturing proceeds more quickly at higher temperatures in alkali,even if the heat-denaturing temperature is not reached). For theembodiment that involves denaturing by elevated temperature (>94° C.),then the denaturing chamber 21 of each cassette will be kept at therequired high temperature, but the remaining chambers will be kept atmuch lower temperatures, which may vary according to the particulartarget for amplification. Nevertheless, the temperature control for thedenaturing chamber should preferably, for flexibility of use, be capableof maintaining any desired temperature likely to be required (i.e. inthe range of ambient temperature to greater than 94° C.).

As previously noted, instead of using coated magnetic beads, the solidtransport medium may be a non-magnetic solid or coated solid in the formof coated particles or sheets. The particles or sheets may then bewithdrawn directly from one chamber and inserted into another bynon-magnetic means. A suitable arrangement for this is shown in FIG. 8.This apparatus is similar to the apparatus of FIGS. 6a-6d, except thatthere is no magnet 27 and a wire frame 29' is provided directly aboveeach cassette 20' and each cassette 20' is provided with a centrallongitudinal slot 50 in the upper wall 34'. The frame carries a holder28', the bottom end of which holds a piece 51 of a paper type transportmedium (e.g. nylon paper). The piece of paper 51 carries the nucleicacid and moves it from chamber to chamber as the holder 28' is drivenalong the frame 29'.

As previously noted, an alternative procedure may be carried out whichthe nucleic acid is bound to a solid anchoring support and remainsstationary, and separate reagent solutions are successively andtemporarily brought into contact with the bound nucleic acid. In such acase, the separate reagent solutions may be reduced to just two, forexample:

A denaturing solution, containing (for example):

a) Specific (e.g. 40 mer) oligonucleotide (e.g. 1 μM)

b) A DNA template;

and

a polymerization solution, containing (for example):

a) A thermolabile polymerase (this may even be added at the beginning ofthe process) (preferably containing 1 unit of polymerase);

b) A mixture of four nucleotides (dCTP, dTTP, dATP, dGTP) (preferably200 μM of each); and

c) MgCl₂ (preferably 1.0 mM-4.0 mM).

The procedure may be carried out in an apparatus of the type shown inFIG. 9 having three separate containers 52, 53, 54. Containers 52 and 54hold solutions of the type indicated above and container 53 contains asupport and anchoring medium for nucleic acid. In the illustratedembodiment, the support and anchoring medium comprises large beads 10',but could alternatively be a coated interior surface of the chamber 53itself. To bring about the required reaction, the sample DNA isintroduced into chamber 53 and binds to the anchoring and supportmedium. Solutions from chambers 52 and 54 are in turn transferred intoand subsequently removed from the central chamber 53 by aspiration viatubing 55 connected to a vacuum system 56. As the solutions areintroduced into chamber 53 from the two adjacent chambers 52 and 54,appropriate conditions are applied (e.g. elevated temperature) and thesolutions are allowed to remain in the chamber 53 for a time appropriatefor the desired reaction.

The sequences amplified by the methods of the invention may be detectedin any suitable way, as will be readily apparent to persons skilled inthe art. Suitable techniques are disclosed, for example, in "MolecularCloning--A Laboratory Manual" by J. Sambrook, E. F. Fristsch and T.Maniatis, CHL Press, New York (the disclosure of which is incorporatedherein by reference).

For example, 25 μl of solution from the denaturing zone may be aspiratedinto an Eppendorf tube as a sample PCR reaction mixture. The PCRreaction mixture may be cleaned up using a chromatography column (e.g. aG50 Sephadex™ column--obtainable from Pharmacia Fine Chemicals, Sweden)and then treated further depending upon the type of labelling employed.

The invention is illustrated in more detail by outlining steps of apreferred embodiment of the process of the invention as it may becarried out in practice.

EXAMPLE 1

Identification of microbes may be carried out by differentmethodologies. This includes isolation of microbes by culturing, ordetecting any specific gene products (proteins) by immunological methods(ELISA). With the discovery of Taq polymerase, the polymerase chainreaction (PCR) is substituting for the conventional identificationmethodologies. PCR technology is quicker, with higher specificity andsensitivity. The main advantage of PCR is that within a matter of 60minutes the main component of the cell nucleic acid can be multiplied toan extent that will take more than 18 hrs to achieve by culturing. Suchidentification involves three steps. These are:

a) Preparation of DNA from the sample (clinical or environmental).

b) Amplification of DNA by PCR.

c) Detection of amplified DNA.

Preparation of DNA (Using Bacterial Cells as an Example)

1. The suspension of bacterial cells is centrifuged at 5,000 rpm in amicrofuge and the supernatant thrown away.

2. The pellet is resuspended in 100 μl of TE buffer at pH 7.0 and step(1) repeated.

3. To the resuspended cell pellet a mixture (200 μl) of 0.2N NaOH and 1%SDS is added, mixed and left on ice/15 minutes.

4. To the above mixture add 150 μl of 3M potassium acetate and invertthree times and leave it on ice.

5. The mixture is microfuged at 10,000 rpm and the suspension istransferred to a new tube and 500 μl of saturated phenol is added andmixed for 3 minutes.

6. The mixture is centrifuged at 10,000 rpm for 5 minutes and the upperlayer is transferred to a new Eppendorf® tube.

7. 500 μl of chloroform is added, mixed and centrifuged at 10,000 rpmfor 5 minutes.

8. The upper aqueous layer is transferred to a new Eppendorf® tube and350 μl of isopropanol is added, mixed and left for 15 minutes.

9. The mixture is microfuged at 15,000 rpm for 15 minutes, thesupernatant is removed and the DNA pellet is dried in a speed vac.

Isothermal Amplification

1. The isolated genomic DNA is bound to Hy-bond® nylon paper.

2. The DNA bound nylon paper is attached to a frame which is moved byrobotic arm. This conveyor belt has a metal strip. The conveyor belt ismade to move by movement of a magnet held external to the containers(FIG. 3).

3. When the DNA bound nylon goes through the alkali container (pH 12.0)the hydrogen bonds between the DNA strands are broken and the bases oneach are exposed.

4. Movement of the conveyor belt takes the nylon paper to theneutralizing container where it is washed by 2×SSC (3M Sodium chlorideand 0.3M Sodium citrate solution at pH 7.0) still maintaining thedenatured state of the DNA strands. During this phase the nylon paper isreturned to neutral pH 7.0.

5. From here the nylon paper is moved to the annealing container wherethe ionic concentration is dropped such that only specific primers withhighly complementary primers will anneal. The high stringency willprevent any nonspecific primers binding or annealing to the exposedstrands.

6. The nylon paper moves to the last container where with the help ofpolymerase, magnesium chloride, dNTP's and polymerase buffer,polymerization takes place.

7. The cycle is repeated by the nylon paper moving to the firstcontainer which is the denaturing container. The new DNA strands formedwill then bind to the nylon paper.

8. At the end of certain number of cycles (approximately 30) there willbe an adequate amount of DNA on the nylon paper and/or in the denaturingchamber.

9. This can be now detected by dot blot using an antibody labeled probeor eluted to carry out further analysis such as sequencing and cloning.

EXAMPLE 2

Two-Chambered Heat Denaturation Procedure The Identification of TargetDNA

An apparatus of the type shown in FIGS. 6a-6d, except containing onlytwo chambers (a denaturing chamber and a polymerization chamber), isprovided. A drive frame suitable for two chambers is also provided formoving a magnet between the chambers. The denaturing chamber containsfreeze dried material consisting of the following:

a) Magnetic beads with specific (40 mer) oligonucleotides (1 μM); and

b) Specific labeled primers (the labeling may be via radioactive biotin,antibody or fluorescent dye) (1 μM).

The polymerization chamber contains a freeze dried material consistingof the following:

a) A thermolabile polymerase (although this may be introduced at thestart of the process, if desired) (1 unit);

b) A mixture of four nucleotides dCTP, dTTP, dATP and dGTP (200 μM ofeach); and

c) MgCl₂ (1.0 mM-4.0 mM).

As a first step, 25 μl of nuclease-free water is added to thepolymerization chamber to cause the contents to dissolve, and a DNAsample in 25 μl of nuclease-free water is added directly into thedenaturing chamber. The denaturing chamber is heated to a temperaturegreater than 94° C. This causes the double stranded DNA to separate intosingle strands.

In the next step, the temperature in the denaturing chamber is reducedto a specific annealing temperature for a suitable time (30 seconds to 1minute). This oligomers attached to the magnetic particles anneal to thecorresponding strand of the DNA. Because initially there are few copies(usually less than 10) of the target DNA strands, only a few of themagnetic beads will carry a duplex of single stranded DNA and a labeledprimer.

The magnet is moved along the drive frame to cause the beads, i.e. boththose which carry the oligomer primers and single stranded DNA and thosewhich carry only the oligomer primers, to exit the denaturing chamberand enter the polymerization chamber. The oligomer primer primes thepolymerization reaction in the presence of the dNTP, MgCl₂ and thepolymerase enzyme. Polymerization thus takes place.

The magnet on the drive frame is then moved back to the startingposition so that the beads exit the polymerization chamber and re-enterthe denaturing chamber. The temperature of this chamber is againincreased to above 94° C. for a period of 30 seconds to 1 minute, andthen the temperature is decreased to the specific annealing temperature.The double stranded DNA carried on the magnetic beads separates into theoriginal single DNA strands and an extended complementary singlestranded DNA, still held on a few magnetic beads. The original DNAstrands will bind to new magnetic beads carrying labeled oligomer.Labeled oligomer present in the denaturing chamber binds to the 3' endof the extended complementary single stranded DNA on the magnetic beads.Although original single stranded DNA could bind to the magnetic beadscarrying extended single stranded DNA, the high concentration of labeledoligomer will overwhelm the low concentration (low number of copies) ofthe original target DNA.

The indicated cycle is repeated 25 to 40 times and the process isfinally stopped with the beads in the polymerization chamber. Thecontents of the polymerization chamber are aspirated into a new 200 μleppendorf tube, washed with low ionic buffer and detected according tothe nature of the labeling, i.e.:

Radio isotope X-ray film

Antibody labeling ELISA

Fluorescence Fluorescence detection.

Various modifications may be made to this process. Firstly, instead ofusing only one type of magnetic bead carrying an oligomer specific toonly one of the template DNA strands, two types of beads may beprovided, one specific to the sense strand and one specific to theanti-sense strand. This may speed up the amplification and allow areduction in the number of the cycles.

Secondly, the oligomer attached to the magnetic bead itself may belabeled and also carry an unlabeled 3' binding primer.

EXAMPLE 3

Two Chamber Heat Denaturation Procedure Synthesis of Single Stranded DNATemplates of a Target DNA for Sequencing

An apparatus of the type shown in FIGS. 3a-3d, except containing onlytwo chambers (a denaturing chamber and a polymerization chamber), isprovided. A drive frame suitable for two chambers is also provided formoving a magnet between the chambers. The denaturing chamber containsfreeze dried material consisting of the following:

a) Magnetic beads with a specific (40 mer) oligonucleotide (1 μM).

The polymerization chamber contains a freeze dried material consistingof the following:

a) A thermolabile polymerase (although this may be introduced at thestart of the process, if desired) (1 unit);

b) A mixture of four nucleotides dCTP, dTTP, dATP and dGTP (200 μM ofeach); and

c) MgCl₂ (1.0 mM-4.0 mM).

In a first step, 25 μl of nuclease-free water is added to thepolymerization chamber to dissolve the contents. A DNA sample in 25 μlof nuclease-free water is introduced directly into the denaturingchamber. The contents of the denaturing chamber are heated to atemperature above 94° C. and the double strands separate into singlestrands.

As a next step (which is eliminated after about 10 cycles), thetemperature is lowered to a specific annealing temperature for 30seconds to 1 minute. The magnetic particles provided with the oligomeranneal to the corresponding single strand of the DNA. Because initiallythere will be few copies (normally less than 10) of the target singlestranded DNA, only a few of the magnetic beads provided with theoligomer will carry a single stranded DNA molecule as a duplex.

The magnet on the drive frame is then moved so that the magnetic beads,i.e. both those carrying the duplex and those carrying only theoligomer, are moved to the polymerization chamber where the oligomeracts as a primer and, in the presence of the DNTP, MgCl₂ and polymeraseenzyme, causes polymerization to take place.

The magnet is then moved to the starting position so that the beadsre-enter the denaturing chamber, where the temperature is increasedabove 94° C. for 30 seconds to 1 minute. The temperature is thendecreased to the specific annealing temperature (as noted above, afterabout 10 cycles this step is eliminated). The DNA strands on themagnetic beads carrying polymerized DNA separate into the originalsingle stranded DNA copy and an extended complementary single strandedDNA on a few of the magnetic beads. After the initial 10 cycles or so,there will no longer be any new magnetic beads carrying availableoligomers, so that these single stranded DNA will be left as unboundstrands.

The cycle is repeated 25 to 40 times and the process is finally stoppedwith the beads positioned in the polymerization chamber. The contents ofthe denaturing chamber are aspirated while it is at 94° C. and is thusready for sequencing.

The extended DNA sequence attached to the magnetic beads will act as atemplate for new priming and hence at the end of each cycle at thedenaturation step the newly synthesized DNA will be separated from themagnetic bead and left in the chamber (asymmetric amplification). Thenumber of beads should be such that it will be able to shorted the timeof amplification and that adequate copies of the newly synthesized DNAstrand with the labeled primers will accumulate for detection.

This procedure may be modified in the following ways.

Firstly, the contents of the polymerization chamber may be alteredslightly so that, in addition to the ingredients mentioned above, itwill also contain one of the dedioxy nucleotides (e.g. ddTTP). At theend of the process, the contents of the denaturing chamber will includesingle stranded copies of the template exhibiting termination withddTTP. This will be adequate to allow analysis of a single nucleotidetract sequence.

Secondly, if a full nucleotide sequence is required, the procedure maybe carried out using four cassettes each carrying one of ddTTP, ddCTP,ddATP and ddGTP.

EXAMPLE 4

Detection of Amplified Sequence

This Example illustrates how amplified sequences may be extracted anddetected following the reactions of the present invention. The followingsteps are carried following PCR in a cassette of the type shown in FIGS.6a-6d.

1. The cover above the denaturing chamber is removed and 25 μl of thereaction mixture is aspirated into an Eppendorf tube.

2. The PCR reaction mixture is cleaned up using a G50 Sphadex™ column(Pharmacia, Fine Chemicals, Sweden).

3. Depending on the type of labelling:

a) For radio labeled probes, the mixture is placed on a nylon paper andautoradiographed for signals.

b) For fluorescence probes, the mixture is diluted in suitable diluentsand read on a fluorescence scanner (Hitachi FMBIO II).

c) For biotinylated probes, the mixture is placed on a nylon paper. Thisis followed by addition of strepavidin conjugated antibody which isfollowed by species specific antibody conjugated with alkalinephosphatase or horse radish peroxidase.

Detection with Biotinylated Primers

1. The nylon filter is transferred from the final wash in SSC to a traycontaining 200 ml of 150 mM NaCl, 50 mM Tris.Cl(pH 7.5). The filters areincubated for 10 minutes at room temperature with gentle agitation.

2. The filter is transferred to a heat sealable plastic bag or to ashallow tray, containing 0.1 ml of phosphate free, azide-free blockingsolution per square centimeter of filter.

3. An enzyme-coupled secondary reagent is added according to themanufacturer's instructions and the bag (if used) is sealed. Usually itis recommended that the secondary reagent be diluted 1:200 to 1:2000 toyield a final concentration of 0.5-5.0 μg/ml.

4. The filter is incubated with an enzyme-coupled secondary reagent for1 hour at room temperature with gentle agitation. The filter istransferred to a tray containing 200 ml of 150 mM NaCl, 50 mM Tris.Cl(pH7.5). The filter is incubated for 10 minutes at room temperature withgentle agitation. This step is repeated three more times using freshNaCl/Tris.Cl solution each time. Add appropriate chromogenic substratesto the filter.

Chromogenic Substances

1. NBT (nitro blue tetrazolium) is prepared. The substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)is converted in situ into a dense blue compound by immuno localisedalkaline phosphatase. 0.5 g of NBT is dissolved in 10 ml of 70%dimethylformamide.

2. BCIP (Bromo chloro-indo)-phosphate is prepared. 0.5 g of BCIPdisodium salt is dissolved in 10 ml of 100% dimethylformamide.

3. Alkaline phosphatase buffer.

4. 66 μl of NBT stock is mixed with 10 ml of alkaline phosphatasebuffer. The mixture is mixed well and 33 μl of BCIP stock is added. Thischromogenic substrate mixture should be used within 30 minutes.

5. Washed nylon filter is transferred to a shallow tray. 0.1 ml ofchromogenic substrate mixture per square centimeter of filter is added.The filter is incubated at room temperature with gentle agitation.

6. The progress of the reaction is monitored carefully. When the bandsare of the desired intensity, the filter is transferred to a traycontaining 200 μl of 0.5 M EDTA (pH 8.0) and 50 ml of phosphate bufferedsaline. The filter is photographed to provide a permanent record.

Alternatively horse radish peroxidase may be used with3,3'-diaminobenzidine, which is converted to a brown precipitate.

Antibodies coupled to HRP (Horse radish peroxidase) or Ap (Alkalinephosphatase) that react with species-specific determinants on primaryantibodies are available from commercial source (Sigma Chemicals, USA)Avidin-conjugated HRP are available from commercial sources as well asbiotinylated primers (Pierce, USA).

The single stranded DNA may be used as a template for DNA sequencing.This approach may be adopted to an application which involves detectingpoint mutations for cancer susceptibility.

While the invention and embodiments thereof have been described indetail above, it will be apparent that various modifications andalterations will be possible without departing from the spirit and scopeof the invention. All such variations and modifications are includedwithin the scope of the present application.

What we claim is:
 1. Apparatus for amplifying a nucleic acid sequence bya process involving a polymerase or ligase chain reaction, which processcomprises repeated cycles of steps including a nucleic acid denaturingstep and a nucleic acid synthesis step, said synthesis step beingcarried out under action of an enzyme, wherein the denaturing step andthe synthesis step are carried out in different denaturing and synthesisreaction zones, respectively, and wherein, during said repeated cycles,the enzyme is maintained in isolation from the denaturing reaction zone,and conditions or reagents required for the denaturing step aremaintained in isolation from the synthesis reaction zone to the extentthat said reactions and conditions do not impede said synthesis reactionsubstantially; said apparatus comprising a first container defining saiddenaturing zone for holding reactants required during said denaturingstep, a second container defining said synthesis reaction zone forholding reactants required during said synthesis step, including saidenzyme, a first and second containers being physically separateentities, a movable solid phase support for binding nucleic acid formedduring said synthesis step, and means for repeatedly moving said solidphase support from said first container to said second container andthen back to said first container for a plurality of cycles until saidamplifying of said nucleic acid sequence is complete, while avoidingtransfer of substantial amounts of reactants between said first andsecond containers.
 2. Apparatus according to claim 1, wherein said meanscomprises a drive mechanism for transporting said solid phase supportsuccessively between said containers.
 3. Apparatus according to claim 2,wherein said solid phase support is magnetic, and wherein said drivemechanism includes a magnet for attracting said solid phase support andmeans for moving said magnet to cause said solid phase support to movebetween said containers.
 4. Apparatus according to claim 2, wherein saidsolid phase support is in the form of a sheet of material, and whereinsaid drive mechanism includes an attachment for holding said sheet andmeans for moving said attachment to cause said sheet to move frombetween said containers.
 5. Apparatus according to claim 1, including aholder, and at least one removable cassette unit removably held by saidholder, said cassette incorporating said containers.
 6. Apparatus forcarrying out a process of amplifying a nucleic acid sequence, involvingrepeated cycles of the steps of denaturing of complementary strands ofnucleic acid, annealing of said nucleic acid with at least onecomplementary primer flanking a target sequence, and nucleic acidpolymerase catalyzed polymerization of a sequence of nucleic acidcomplementary to said target sequence extending from said primer(s)using the target sequence as a template for polymerization, saidapparatus comprising:a plurality of containers, each being physicallyseparate entities, for containing liquids forming reaction zones fordenaturing, annealing and polymerization of nucleic acids, a solidsupport for immobilizing said nucleic acid and synthesized nucleic acid,and means for moving said solid support from one of said containers toanother in a sequence appropriate for effecting a polymerase chainreaction, wherein at least a container provided for said synthesis stepis provided with a heater controlled to prevent liquids in saidcontainer from reaching a temperature at which activity of thermolabileDNA polymerase held in said container is destroyed.
 7. A cassette unitfor carrying out a process involving amplifying a nucleic acid sequenceby a polymerase or ligase chain reaction, said process involvingrepeated cycles of steps including a nucleic acid denaturing step and anucleic acid synthesis step, said synthesis step being carried out underaction of an enzyme, wherein the denaturing step and the synthesis stepare carried out in different denaturing and synthesis reaction zones,respectively, and wherein, during said repeated cycles, the enzyme ismaintained in isolation from the denaturing reaction zone, andconditions or reagents required for the denaturing step are maintainedin isolation from the synthesis reaction zone to the extent that saidreactions and conditions do not impede said synthesis reactionsubstantially; said cassette unit comprising an enclosed housing havingat least two internal chambers, that are physically separate entities,for holding liquid reactants and maintaining said liquid reactantsseparate from each other and a solid support for nucleic acid providedin one said container, said housing including at least one passageallowing said solid support to be brought successively and temporarilyinto contact with said liquid reactants from said at least twocontainers.
 8. A cassette unit according to claim 7, wherein said solidsupport comprises beads that may be moved through said passage betweensaid at least two containers.
 9. A cassette unit according to claim 7,wherein said housing has an upper wall provided with at least oneopening covered by a self-sealing pierceable diaphragm.